Upload
others
View
0
Download
0
Embed Size (px)
Citation preview
b i o c h e m i c a l p h a r m a c o l o g y 7 2 ( 2 0 0 6 ) 1 0 1 0 – 1 0 2 1
avai lable at www.sc iencedi rec t .com
journal homepage: www.e lsev ier .com/ locate /b iochempharm
Inhibition of pro-inflammatory markers in primarybone marrow-derived mouse macrophages bynaturally occurring flavonoids: Analysis of thestructure–activity relationship
Mònica Comalada a,*, Isabel Ballester a, Elvira Bailón a, Saleta Sierra b,Jordi Xaus b, Julio Gálvez a, Fermı́n Sánchez de Medina a, Antonio Zarzuelo a
aDepartment of Pharmacology, School of Pharmacy, University of Granada,
Campus Universitario ‘‘La Cartuja’’ s/n, 18071-Granada, SpainbDepartment of Immunology and Animal Sciences, Puleva Biotech, Granada, S.A., Spain
2 3
atory effect.
with the double bond
highest anti-inflamm
a r t i c l e i n f o
Article history:
Received 8 May 2006
Accepted 18 July 2006
Keywords:
Flavonoids
Inflammation
Macrophages
Structure–activity analysis
iNOS
TNF
a b s t r a c t
Flavonoids possess several biological/pharmacological activities including anticancer, anti-
microbial, antiviral, anti-inflammatory, immunomodulatory and antioxidant. The aim of
this study was to evaluate the effect of flavonoids on macrophage physiology. For this
purpose we selected some flavonoids belonging to the most common and abundant groups
(flavonols—quercetin and kaempferol; flavones—diosmetin, apigenin, chrysin and luteolin;
isoflavones—genistein and daidzein and flavanones—hesperetin). We decided to use pri-
mary bone marrow-derived macrophages (BMDM) as cellular model, since they represent a
homogenous, non-transformed population of macrophages that can be stimulated in vitro to
proliferate by macrophage colony-stimulating factor (M-CSF) or activated by LPS. In this
regard, we demonstrated that most of the flavonoids assayed reduce macrophage M-CSF-
induced proliferation without affecting cellular viability. Moreover, some flavonoids also
inhibit TNFa production as well as iNOS expression and NO production in LPS-activated
macrophages, an effect that has been associated with the inhibition of the NF-kB pathway.
We also found that luteolin and quercetin are able to stimulate the expression of the anti-
inflammatory cytokine IL-10 at low concentrations (
b i o c h e m i c a l p h a r m a c o l o g y 7 2 ( 2 0 0 6 ) 1 0 1 0 – 1 0 2 1 1011
subclasses are flavones, flavonols, isoflavones and flavanones.
The first two are the most commonly occurring flavonoids in
plants, while flavanones are specially abundant in citrus fruits
[1,2]. The isoflavones daidzein and genistein and the flavones
apigenin and luteolin were recently identified as the main
flavonoids accumulated in soybean cotyledons [3,4].
Flavonoids possess various biological/pharmacological
activities including antioxidant, antitumour, antiangiogenic,
anti-inflammatory, antiallergic and antiviral properties [5]. Of
these biological activities, the anti-inflammatory capacity of
flavonoids has long been used in Chinese medicine and the
cosmetic industry as crude plant extracts. Moreover, some
flavonoids have been found to inhibit chronic inflammation in
several experimental animal models [6–9]. Current clinical
approaches to the treatment of inflammation typically focus on
the inhibition of pro-inflammatory mediator production, non-
steroidal anti-inflammatory drugs like ibuprofen being a good
example. Thus, flavonoids are promising anti-inflammatory
drugs that warrant a comprehensive evaluation of its clinical
potential as a putative new class of anti-inflammatory agents.
The inflammatory response involves the sequential release
of mediators and the recruitment of circulating leukocytes,
which become activated at the inflammatory site and release
further mediators. The persistent accumulation and activa-
tion of leukocytes is a hallmark of different chronic diseases,
such as inflammatory bowel disease [10], rheumatoid arthritis
[11,12] and septic shock [13]. The capacity to proliferate
continuously or a reduction of the clearance of activated cells
by apoptosis in the inflammatory site also allows the
perpetuation of the inflammatory response.
Macrophages perform essential functions in the organism
by acting as regulators of homeostasis and as effector cells in
infection, wounding and tumor growth [14]. According to the
local needs, resident macrophages either continue proliferat-
ing in the presence of M-CSF, the specific growth factor for
these cells [15], or become activated and perform their
specialized functions [16]. Macrophage activation is charac-
terized by a series of biochemical and morphological mod-
ifications that allow these cells to perform their professional
functions [14,16]. LPS is an outer membrane component of
Gram-negative bacteria and a potent activator of monocytes
and macrophages. LPS binds to surface toll like receptor 4
(TLR4), triggering the secretion of a variety of inflammatory
products, such as tumour necrosis factor-a (TNFa) and
interleukin-1b (IL-1b), as well as high amounts of nitric oxide
(NO), which contribute to the pathophysiology of septic shock
and other immune diseases [17,18]. Production and release of
inflammatory mediators and cytokines by LPS depends on
inducible gene expression mediated by the activation of
different transcription factors, such as NF-kB [19].
There have been several in vitro studies to investigate the
inhibitory activity of flavonoids on NO and cytokine production
in different macrophage cell lines, such as RAW 264.7 [18,20–23]
and J774.1 [24]. However, the use of immortalized cell lines does
not allow the observationofphysiologic effectsonproliferation-
differentiation since the biochemical machinery involved in
these processes is disturbed. Here we used primary culture of
macrophages obtained from bone marrow, since they represent
a homogeneous, non-transformed population of macrophages
that can be stimulated in vitro to induce proliferation,
differentiation or apoptosis, but also activated to analyze the
expression of inflammatory cytokines [25].
The goal of the present study was to test the effects on
proliferation and survival as well as the anti-inflammatory
properties of different flavonoid compounds (Table 1), to study
the structure–activity relationship and to explore some
mechanistic aspects of the effects observed. Specially, we
focused on the NF-kB pathway, because of its well-character-
ized involvement in the inflammatory response mediated by
macrophages. We selected these nine flavonoids because they
are the most common in the human diet and each flavonoid is
representative of the different substitutions and structural
forms typical of the different flavonoid subclasses (see
differences in Table 1). In this regard, we have confirmed the
previously reported involvement of the NF-kB pathway in the
anti-inflammatory effect of flavonoids but, for the first time, we
have observed a new potential mechanism exerted by some
flavonoids to modulate the inflammatory response such, i.e. the
induction of IL-10 expression exerted by luteolin and quercetin.
2. Materials and methods
2.1. Reagents
Flavonoids were purchased from Extrasynthèse (Genay,
France), except genistein and luteolin, which were obtained
from Sigma (Madrid, Spain), along with all other reagents
unless stated otherwise. In some experiments, we used
sulfasalazine as a positive control of an anti-inflammatory
drug or a NF-kB-activation inhibitor [26,27]. Flavonoids were
diluted in DMSO stock solutions prior to addition to cell culture
medium. The final concentration of DMSO never exceeded
0.1% (v/v).
2.2. Macrophage culture
BMDM were isolated from 6-week-old Balb/c mice (Laboratory
Animal Service of the University of Granada) as previously
described [28]. Mice were killed by cervical dislocation, and
both femurs were dissected free of adherent tissue. The ends
of the bones were cut off, and the marrow tissue was flushed
by irrigation with culture medium. The marrow plugs were
dispersed by passing a 25-gauge needle through them, and the
cells were suspended by vigorous pipetting and washed. Cells
were cultured in 150 mm Petri dishes with 40 ml of DMEM
containing 20% FBS and 30% L-cell-conditioned medium as a
source of macrophage colony-stimulating factor (M-CSF) [25].
Cells were incubated at 37 8C in a humidified 5% CO2atmosphere. After 6 days of culture a homogeneous popula-
tion of adherent macrophages was obtained (>99% Mac-1+; BD
Pharmingen, Heidelberg, Germany) (data not shown). To
render cells quiescent, when macrophages were 80% con-
fluent they were deprived of L-cell-conditioned medium for
16 h before carrying out the proliferation assay.
2.3. Proliferation assay
Cell proliferation was measured by [3H]-thymidine incorpora-
tion as previously described [29], with minor modifications.
b i o c h e m i c a l p h a r m a c o l o g y 7 2 ( 2 0 0 6 ) 1 0 1 0 – 1 0 2 11012
Table 1 – Structural features of the flavonoids tested
Chemical formula Name Substitution
5 7 30 40
Flavonols
Kaempferol OH OH H OH
Quercetin OH OH OH OH
Flavones
Apigenin OH OH H OH
Chrysin OH OH H H
Diosmetin OH OH OH OCH3Luteolin OH OH OH OH
Isoflavones
Daidzein OH H H OH
Genistein OH OH H OH
Flavanones
Hesperetin OH OH OH OCH3
Quiescent cells (105) were incubated in 24-well plate in the
presence or absence of the indicated flavonoids (25, 50 and
100 mM) or vehicle (0.1%, v/v DMSO) for 1 h before the addition
of M-CSF (900 U/ml) and were incubated for 24 h. Then,
macrophages were pulsed with [3H]-thymidine (1 mCi/well) for
6 h at 37 8C. After this period cells were fixed in ice-cold 70%
trichloroacetic acid and solubilized in 1% SDS and 0.3 M NaOH
at room temperature. Radioactivity was counted by liquid
scintillation counter. [3H]-Thymidine uptake was expressed as
the percentage of the maximum uptake (M-CSF alone). All
assays were performed in triplicate and the results are
expressed as mean � S.D.
2.4. Determination of TNFa and IL-10 concentration
Cells were cultured in 24-well plate in the presence or absence
of different flavonoid concentrations as indicated for 1 h
before the stimulation with LPS (10 ng/ml). Supernatants were
obtained 24 h after and frozen at �80 8C until ELISA analysis.Cytokine production was measured with commercial murine
ELISA kits (CytosetsTM, Biosource International, Nivelles,
Belgium) following the manufacturer’s protocol.
2.5. Protein extraction and Western blot analysis
Macrophages were washed with PBS and homogenized in cold
lysis buffer containing 1% Igepal CA-630, 20 mM HEPES–Na pH
7.5, 10 mM EGTA, 40 mM b-glycerophosphate, 25 mM MgCl2,
2 mM sodium orthovanadate and freshly added protease
inhibitors (phenylmethylsulfonyl fluoride, aprotinin, leupeptin,
1,10-phenanthroline). The protein contentwasmeasuredbythe
bicinchoninic acid assay [30], using bovine serum albumin as
standard. Samples were boiled for 4 min in Laemmli buffer,
then 100 mg (iNOS) or 65 mg (phospho-IkB-a) were separated by
7% and 10% SDS-PAGE, respectively. The nitrocellulose mem-
branes wereblocked for at least 1 h at roomtemperature in Tris-
buffered saline–0.1% Tween-20 (TBS-T) containing 5% (w/v)
nonfat dry milk and then incubated with TBS-T containing BSA
5% and the primary antibody at 4 8C overnight. The dilutions of
antibodies used were: 1:3000 for iNOS (Transduction Labora-
tories, BD Biosciences, Madrid, Spain) and 1:1500 for phospho-
IkB-a (Cell Signaling Technology, Beverly, MA). A primary
antibody against a-actin Sigma (Sigma, Madrid) was used as
loading control. After three washes of 5 min with TBS-T,
peroxidase-conjugated anti-rabbit or anti-mouse IgG was used
as secondary antibody. Then, enhanced chemiluminiscence
(Perkin-ElmerTM, Life Sciences, Boston, USA) detection was
performed.
2.6. Determination of NO production
NO production was estimated by measuring nitrate/nitrite in
the cell culture media. Macrophages were cultured in DMEM
without phenol-red (Invitrogen) to avoid interference with the
Griess absorbance at 550 nm. Samples were stored at �80 8Cuntil assayed. Nitrate was converted to nitrite with Zea mays
nitrate reductase (Calbiochem). Reduced samples were incu-
bated with an equal volume of Griess reagent, and the
b i o c h e m i c a l p h a r m a c o l o g y 7 2 ( 2 0 0 6 ) 1 0 1 0 – 1 0 2 1 1013
absorbance at 550 nm was measured. The total nitrate/nitrite
concentration was determined by comparison with a standard
curve.
2.7. Analysis of gene expression by RT-PCR
Total cellular RNA was isolated by single-step, guanidium
thiocyanate–phenol–chloroform extraction with the TRIzol1
Reagent (Gibco-BRL, USA) according to the manufacturer’s
instructions. RNA (2 mg) from each sample was reverse
transcribed into complementary DNA (cDNA) using reverse
transcriptase (First-Strand cDNA Synthesis Kit, Amersham
Biosciences) and following the instructions as indicated. The
primer sequences were: IL-10 (forward 50-TCCTTAATGCAG-
GACTTTAAGGG-30, reverse 50-GGTCTTGGAGCTTATTAAAAT-
30), b-actin (forward 50-TGGAATCCTGTGGCATCC-30, reverse 50-
AACGCAGCTCAGTAACAGTCC-30). The polymerase chain
reaction was performed in a 25 ml volume containing 3 ml of
RT product (cDNA) and 22 ml PCR master mix: 10� buffer, 1 UTaq DNA polymerase (Biotherm Genecraft, Germany), 0.2 M of
each dNTP (Roche, Germany) and 1 mM concentration of each
primer. For each primer pair, control experiments were
performed to determine the range of cycles in which a given
amount of cDNA would be amplified in a linear fashion: 28
cycles for both IL-10 and b-actin. Semi-quantitative analyses
of ethidium bromide-stained DNA gels (2% agarose) were
performed with Scion Image (Scion Corporation, USA). The
data were normalized to the constitutively expressed b-actin
gene transcript levels.
2.8. Statistical analysis
All results are expressed as mean � S.D. Differences amongmeans were tested for statistical significance using a one-way
analysis of variance and post hoc least significance test. The
concentration–response curves were fitted with a sigmoidal
logistic equation using the Origin 5.0 software (Microcal,
Northampton, MA), which provided the calculation of the EC50.
All other statistical analyses were carried out with the
Statgraphics 5.0 software package (STSC, MD), with statistical
significance set at p < 0.05.
Fig. 1 – Effect of flavonoids on M-CSF-dependent
proliferation of BMDM. Macrophages were incubated in
the presence of 900 U/ml of M-CSF in 24-well plate with or
without flavonoids (25 and 50 mM). (A) Flavonols:
kaempferol and quercetin; (B) flavones: apigenin, chrysin,
diosmetin and luteolin; (C) isoflavones: daidzein and
genistein; and a flavanone: hesperetin. Each assay was
performed in triplicate, and the results are represented as
mean W S.D. of the percentage (%) of thymidine uptake in
comparison to non-flavonoid treated macrophages
(*p < 0.05, **p < 0.01).
3. Results
3.1. Effects of flavonoids on M-CSF–macrophageproliferation and viability
For this study we used macrophages obtained from bone
marrow cultures, primary cells that can proliferate efficiently in
response to M-CSF, the main growth factor used by macro-
phages. To determine the involvement of flavonoid compounds
in the response to M-CSF, we used [3H]-thymidine incorporation
as an indicatorof cell proliferation, since wehave demonstrated
previously that this is a valid method that correlates with an
increase in the number of macrophages [29]. On the basis of
these former studies the concentration of 900 U/ml of M-CSF
was selected to induce macrophage proliferation. When flavo-
nols (kaempferol and quercetin) are added to the culture super-
natants, macrophage proliferation is inhibited significantly
(Fig. 1A). This effect is concentration dependent, and macro-
phage proliferation is completely inhibited at a concentration of
50 mM of flavonols. Additionally, the effect of vehicle (0.1%
DMSO) was evaluated on the proliferation of macrophages and
no inhibitory effect was observed (Fig. 1A).
Moreover, the proliferative response of bone marrow
macrophages to M-CSF is markedly reduced in a concentra-
tion-dependent manner in the presence of flavones (25 and
50 mM), with diosmetin and luteolin being the most efficient
compounds (Fig. 1B). On the other hand, isoflavones have a
b i o c h e m i c a l p h a r m a c o l o g y 7 2 ( 2 0 0 6 ) 1 0 1 0 – 1 0 2 11014
Fig. 2 – Effect of flavonoids: Gen (genistein), Quer (quercetin)
and Lut (luteolin) on LPS-induced TNFa release from
mouse macrophages. Cells were pretreated for 1 h with
vehicle (DMSO: 0.1%, v/v) or the flavonoid (1, 10, 25, 50,
100 mM) and then stimulated with LPS (10 ng/ml) for 24 h.
The medium was collected and analyzed by ELISA as
described in Section 2. Results are expressed as the
mean W S.D. of percentage of TNFa secretion in
comparison to non-flavonoid treated macrophages (n = 3).
The EC50 values are 10.0 W 0.3 mM (luteolin), 20.0 W 0.6 mM
(quercetin) and 30.0 W 0.9 mM (genistein).
lower impact on macrophage proliferation (Fig. 1C), since only
genistein shows a significant inhibition at the concentration of
50 mM. Finally, treatment of macrophages with hesperetin
results in inhibition of proliferation only at the concentration
of 100 mM (50% of the control response, Fig. 1C). These results
suggest that flavones and flavonols seem to be more efficient
than isoflavones or flavanones as inhibitors of M-CSF-induced
macrophage proliferation.
Since previous reports indicate that flavonoids may induce
apoptosis in different cultured cells [31–33], we decided to
examine the influence of flavonoid compounds on macrophage
viability by crystal violet staining. In all flavonoid groups, none
of the compounds tested reduces significantly macrophage
viabilityat25 or50 mM (data not shown). Onlyquercetin, chrysin
and luteolin show a slight viability reduction, not higher that
20% in any case, at 100 mM (data not shown).
3.2. Effect of flavonoids on TNFa release by LPS-activatedmacrophages
We also determined the effects of flavonoid compounds on
macrophage activation induced by LPS. To this end, cultured
mouse macrophages were pretreated with flavonoids (25–
50 mM) and the release of TNFa to culture medium measured
by ELISA. Virtually all flavonoids tested inhibit LPS-induced
TNFa secretion with variable success (Table 2). Thus daidzein
and chrysin show only a slight effect on TNFa release (60% inhibition)
(Table 2).
The inhibitory effect of the three most active inhibitors
(quercetin, luteolin and genistein) was further analyzed by
assaying lower concentrations of these flavonoids on TNFa
secretion (Fig. 2). Genistein inhibits progressively TNFa
secretion without achieving total blockade, whereas quercetin
and luteolin show a more sigmoidal tendency. The EC50 values
obtained with luteolin and quercetin were 10.0 � 0.3 mM and20.0 � 0.6 mM, respectively, whereas the corresponding valuefor genistein was 30.0 � 0.9 mM. These results confirm thatluteolin, quercetin and genistein inhibit TNFa secretion in a
concentration-dependent manner, in that order of potency
(Fig. 2).
3.3. Effect of flavonoids on iNOS expression and NOrelease by LPS-activated macrophages
NO is involved in various biological processes including
inflammation [34,35]. Since LPS induces the expression of iNOS
in macrophages, and the synthesis of iNOS protein correlates
with NO production [36], we analyzed the effect of flavonoids on
iNOS expression induced by LPS treatment (10 ng/ml) for 6 h
(Fig. 3). Our results show that the flavonol quercetin and the
flavones apigenin, luteolin and diosmetin downregulate iNOS
expression at low concentrations (�50 mM) (Fig. 3), while higherdoses (100 mM) of kaempferol (flavonol) or chrysin (flavone) are
requiredto obtain an inhibitory effect (Fig. 3A and B). Finally, the
flavanone hesperetin and the isoflavones genistein and
daidzein fail to exert any inhibitory effect on iNOS expression
even at 100 mM (Fig. 3C and D).
To confirm these results NO release was also measured
indirectly by the quantification of nitrite concentration in the
b i o c h e m i c a l p h a r m a c o l o g y 7 2 ( 2 0 0 6 ) 1 0 1 0 – 1 0 2 1 1015
Fig. 3 – Effects of (A) flavonols, (B) flavones, (C) isoflavones
and (D) flavanones, on LPS-induced iNOS expression in
mouse macrophages. Cells were pretreated with the
flavonoids for 1 h and then incubated with LPS (10 ng/ml)
for 6 h. Cells lysates were processed and iNOS expression
was analyzed by Western blot. Densitometric analysis of
the westerns blots was performed and the values obtained
are represented in the figure as the normalized band
intensity (iNOS/actin) and referred as the LPS-stimulated
value (1.00). These experiments were performed at least
three times. Quer (quercetin), Kaemp (kaempferol), Api
(apigenin), Chry (chrysin), Diosm (diosmetin), Lut
(luteolin), Gen (genistein), Daid (daidzein), Hesp
(hesperetin).
Table 3 – Effects of flavonoids on LPS-induced NO releasein bone marrow-derived macrophages (%inhibi-tion W S.D.)
Flavonoid Concentration (mM) NO (%inhibition)
Kaempferol 25 28.1 � 6.550 44.3 � 4.3*
Quercetin 25 62.3 � 5.6**
50 85.4 � 7.7**
Apigenin 25 42.2 � 3.8*
50 57.6 � 5.5**
Chrysin 25 11.2 � 4.650 27.9 � 7.9
Diosmetin 25 39.7 � 2.1*
50 58.4 � 0.9**
Luteolin 25 74.8 � 6.6**
50 91.0 � 10.1**
Daidzein 25 17.1 � 1.950 10.5 � 7.1
Genistein 25 14.8 � 6.750 31.4 � 8.2
Hesperetin 25 20.0 � 3.350 19.1 � 4.5
* p < 0.05; vs. non-flavonoid treated LPS-activated macrophages.** p < 0.01; vs. non-flavonoid treated LPS-activated macrophages.
LPS-activated macrophages in presence of the tested flavo-
noids. As resumed in Table 3, again, the flavonol quercetin and
the flavones apigenin, luteolin and diosmetin were able to
inhibit the release of macrophages in a significant way at low
concentrations (25–50 mM) while the flavonol kampferol only
was able to do it at 50 mM concentration. No effect was observed
by the other flavonoids tested on NO production (Table 3).
3.4. Effect of flavonoids on LPS-induced IkB-aphosphorylation
Because both iNOS and TNFa are genes regulated by the NF-kB
transcription factor [37,38], we decided to study the implica-
tion of this pathway as a potential mechanism mediating the
effect of the selected flavonoids. Under quiescent conditions,
NF-kB is sequestered in the cytosol bound to the inhibitory
protein IkB-a. Exposure of cells to LPS triggers phosphorylation
cascades that ultimately lead to phosphorylation and degra-
dation of IkB-a. Once IkB-a dissociates from the complex, NF-
kB translocates into the nucleus where binding to specific DNA
motifs in the promoter region occurs, leading to increased
gene transcription. First, to determine the involvement of IkB-
a phosphorylation in LPS macrophage activation, we used the
NF-kB inhibitor sulfasalazine. Exposure of macrophages to LPS
(10 ng/ml) increases IkB-a phosphorylation on serine 32 as
determined by Western blot (Fig. 4A). Phosphorylation of IkB-a
by LPS is partially abolished in the cells preincubated with
100 mM of sulfasalazine (Fig. 4B). Moreover, the treatment with
sulfasalazine also inhibits iNOS expression after 6 h of LPS
stimulation (Fig. 4C).
Pretreatment of the cells with quercetin reduces IkB-a
phosphorylation significantly at 10 mM and specially at 50 mM
b i o c h e m i c a l p h a r m a c o l o g y 7 2 ( 2 0 0 6 ) 1 0 1 0 – 1 0 2 11016
Fig. 4 – Effect of sulfasalazine treatment in LPS-activated
macrophages. (A) LPS-induced IkB-a phosphorylation in
mouse macrophages. Quiescent cells were stimulated
with LPS (10 ng/ml) at different time points. Total cell
lysates were processed by SDS-PAGE and membranes
blotted with an IkB-a-P antibody. (B) Sulf (sulfasalazine)
inhibits LPS-induced IkB-a phosphorylation at 100 mM.
Sulfasalazine was added 1 h before cells were stimulated
with LPS (10 ng/ml) for 10 min. (C) Sulfasalazine 100 mM
totally inhibits iNOS expression in mouse macrophages.
Densitometric analysis of the westerns blots was
performed and the values obtained are represented in the
figure as the normalized band intensity (IkB-a-P or iNOS/
actin) and referred as the LPS-stimulated value (1.00).
These results represent one of three independent
experiments with similar results.
Fig. 5 – Effect of the flavonols quercetin and kaempferol (A),
the flavones apigenin, chrysin, diosmetin and luteolin (B),
the isoflavone genistein (C) and the flavanone hesperetin
(D) on LPS-induced IkB-a phosphorylation in mouse
macrophages. Cells were pretreated with the flavonoids
for 1 h and then stimulated with LPS (10 ng/ml) for 10 min.
The cellular lysates were processed and phosphorylated
IkB-a levels analyzed by Western blot. Densitometric
analysis of the westerns blots was performed and the
values obtained are represented in the figure as the
normalized band intensity (IkB-a-P/actin) and referred as
the LPS-stimulated value (1.00). These experiments were
performed in triplicate with similar results.
(Fig. 5A). Similarly, apigenin, diosmetin and luteolin at 50 mM
show inhibitory effects on IkB-a phosphorylation (Fig. 5B),
while kaempferol and chrysin have only a slight effect (Fig. 5A
and B). Finally, the isoflavones genistein (Fig. 5C) and daidzein
(data not shown) and the flavanone hesperetin (100 mM) do not
modify IkB-a phosphorylation at all (Fig. 5D). Thus there is a
reasonable correlation with iNOS downregulation and IkB-a
phosphorylation in the flavonoids tested. These findings
suggested to us that the inhibition of NF-kB pathway could
be involved in the mechanism of action of at least some of the
active flavonoids by inhibiting IkB-a phosphorylation.
3.5. Luteolin and quercetin induce IL-10 secretion by LPS-activated macrophages
Finally, because the inflammatory process may be inhibited
also by the production of immunomodulatory cytokines such
as IL-10 or TGF-b [39,40], the effect of the most active
flavonoids on IL-10 secretion was investigated. To this end
macrophages, which are the main source of the anti-
inflammatory cytokine IL-10 [41], were pretreated with the
test compounds and then activated with LPS. The inhibitor of
NF-kB sulfasalazine was also included as a reference drug
(Fig. 6A). Macrophages express IL-10 mRNA after 6 h of LPS
stimulation (Fig. 6). While the control drug sulfasalazine does
not change IL-10 expression at 100 mM, the addition of
apigenin (10, 25 and 50 mM) (Fig. 6) to the cell media before
LPS stimulation inhibits mRNA expression in a concentration-
dependent manner. This was the case with most other
b i o c h e m i c a l p h a r m a c o l o g y 7 2 ( 2 0 0 6 ) 1 0 1 0 – 1 0 2 1 1017
Fig. 6 – IL-10 m RNA expression in macrophages treated
with apigenin, sulfasalazine (A), quercetin or luteolin (B).
Expression was assessed by semi-quantitative RT-PCR.
Densitometric analysis of the PCR bands was performed
and the values obtained are represented in the figure as
the normalized band intensity (IL-10/actin) and referred as
the LPS-stimulated value (1.00). The gels shown are
representative of at least triplicate experiments.
flavonoids tested, although to different degrees (data not
shown). Conversely, quercetin and luteolin increase IL-10
mRNA levels at low concentrations (1–10 mM) (Fig. 6B). This
increase is also observed at the protein level (Fig. 7). However,
the effect of both flavonoids disappears at higher concentra-
tions, and luteolin actually inhibits IL-10 secretion at 100 mM
(Fig. 7).
Fig. 7 – Effects of quercetin, luteolin and sulfasalazine on LPS-in
(DMSO: 0.1%, v/v) (not shown) or the indicated flavonoid (A) and
with LPS (10 ng/ml) for 24 h and medium was collected and ana
performed in triplicate and the results are expressed as the me
control LPS without flavonoid).
4. Discussion
Primary macrophages proliferate in the presence of a specific
growth factor, M-CSF, and when they become activated by
stimuli such as LPS, they stop proliferating and adopt an
activated phenotype, characterized by the expression of early
cytokines, such as TNFa and IL-1b, and NO production, as well
as distinct morphological changes [42]. Moreover, activated
macrophages exhibit a delayed secretion of the anti-inflam-
matory cytokine IL-10, which has the potential to inhibit the
immune response in vivo [43]. We decided to use primary
cultures of macrophages as experimental system because they
constitute a better model than immortalized cell lines for the
characterization of the mechanisms involved in macrophage
proliferation, survival and activation [42].
This report describes the effect of several groups of
flavonoids in these different macrophage functions. The
selection of the flavonoids assayed in this work was based on
their prominent presence in foodstuff and hence in the human
diet, as well as on their chemical diversity, which allows us to
delineate the structural determinants involved in their phar-
macological activity (Tables 1 and 4). Thus four different groups
were included in the study: flavonols (kaempferol, quercetin),
flavones (apigenin,chrysin,diosmetinand luteolin), isoflavones
(daidzein, genistein) and a flavanone (hesperetin).
Most of the flavonoids tested have significant antiprolifera-
tive effects on M-CSF-activated macrophages, but flavones and
flavonols are clearly more efficient than isoflavones or flava-
nones in this regard. In this case the iso position of the B ring or
the absence of a double bond at C2–C3 seems to be correlated
negatively with the antiproliferative activity among the com-
pounds tested (Table 4).M-CSF initiates a mitogenic response by
binding to its receptor and thereby activating the receptor’s
intrinsic tyrosine kinase activity and initiating signaling via
multiple effector-mediated pathways [15]. Since some of the
duced IL-10 secretion. Cells were pretreated with vehicle
sulfasalazine (B). After 1 h, macrophages were incubated
lyzed as described in Section 2. Experiments were
an W S.D. of IL-10 secretion (*p < 0.05, **p < 0.01; respect to
b i o c h e m i c a l p h a r m a c o l o g y 7 2 ( 2 0 0 6 ) 1 0 1 0 – 1 0 2 11018
Table 4 – Overview of flavonoid effects on macrophage physiology and SAR analysis
Group Flavonoid Prolif TNFa iNOS/NO IkB-a-P IL-10
Flavonols Kaempferol +++ ++ �/+ � �Quercetin +++ +++ +++/+++ +++ +++
Flavones Apigenin ++ ++ +++/++ ++ �Chrysin ++ + �/� � �Luteolin +++ +++ +++/+++ +++ +++
Diosmetin +++ ++ ++/++ ++ �
Isoflavones Daidzein � � �/� � �Genistein + ++ �/� � �
Flavanones Hesperetin + ++ �/� � �
Structural motives 2-B ring + � + + +C2 C3 + � + + +30,40-OH ? + � + +N8 OH � + + + +3-OH ? � � ? �
The magnitude of the effect is classified semi-quantitatively based on maximal effect. Inhibition is shown as +/++/+++ except for the effect of
IL-10, where +++ denotes enhancement and � denotes inhibition. Absence of effect is shown as �. The contribution of the structural motives isshown as + (positive), � (negative), � (irrelevant) or ? (unknown).
flavonoids used in this work are tyrosine kinase inhibitors, it is
likely that this property may account for the antiproliferative
effects [44]. The NF-kB pathway is involved in other cellular
functions in addition to its proinflammatory actions, including
cellular survival and proliferation [45]. However, in our experi-
mental model we can discard this possibility because although
flavonoids inhibit theM-CSF–macrophage proliferation, they do
not affect the viability of these cells at the concentrations used
(datanotshown).Moreover,NF-kBdoesnotseemtoplayarolein
macrophage proliferation, since sulfasalazine, at the concen-
tration used, does not inhibit M-CSF–macrophage proliferation
(data not shown).
When we analyzed the effects of flavonoids on macrophage
activation, we observed different responses depending on the
activation marker analyzed (TNFa and iNOS/NO). Thus the
flavonoids with 30 and 40 hydroxylations (quercetin and
luteolin) show the highest degree of inhibition of TNFa,
followed by those with only one hydroxyl group on the B ring
(genistein, kaempferol, apigenin, diosmetin and hesperetin),
regardless of the presence of a double bond in C2–C3, 3-
hydroxylation or iso position of the B ring. Absence of hydroxyl
groups in the B ring virtually abolishes this inhibitory activity.
On the other hand, when the effects on iNOS and NO release
are considered, the requirements differ significantly, since
isoflavones and flavanones are essentially ineffective, point-
ing to a determinant role of the position of the B ring and the
insaturation of the C2–C3 bond. Although Western blotting is
only a semi-quantitative technique, flavones appear to be
more active than flavonols. Moreover, NO quantification
confirmed the results obtained with the iNOS expression
analysis. Among flavones, at least three hydroxyl groups are
required to exert a substantial effect (apigenin, luteolin,
diosmetin), while flavonols seemingly require an extra
hydroxyl (quercetin). Similar findings for these flavonoids
have been described in LPS-treated RAW 264.7 cells [18,46].
However, the structural requirements are different from those
corresponding to inhibition of antigen-triggered proliferative
response in murine and human T cells, in which flavones are
more active than flavonols and flavanones [47]. In our
experimental model we could not make this generalization
regarding flavonoid groups, since flavonols such as quercetin
and flavones such as luteolin were equally active. Of note, our
results do not support the relevance of the 3-OH (Table 4),
which has been previously found to play an important role in
other flavonoid properties such as antioxidative activity (see
below) or the inhibition of DNA topoisomerases described by
Constantinou et al [48].
Although the signaling pathways that lead to induction of
both iNOS and TNFa are very similar, a number of differences
have been described which may account for the observed
differences [27,49]. LPS signaling in macrophages involves a
series of phosphorylation events leading to transcription
factor activation and increased cytokine and iNOS production.
Some of the pathways implicated comprise those involving
the Src-family tyrosine kinases, the serine/threonine kinases
protein kinase A and C, mitogen-activated protein kinase
(MAPKs) and protein kinase B/Akt, and the NF-kB pathway.
Certain flavonoids have been reported previously to inhibit
protein kinase C [50], tyrosine kinase [51], and phospholipases
A2 and C [52,53]. Another possible mechanism includes a
downregulation of iNOS indirectly by inhibition of eicosanoid
production through the COX/LOX pathway [54]. We have
focused on the effect of flavonoids on the NF-kB pathway.
Other authors have previously documented the inhibition of
IkB-a phosphorylation by flavonoids [51]. However, it should
be noted that the correlation between interference with the
NF-kB pathway and downregulation of macrophage activation
is not perfect, and it correlates better with iNOS expression
than with TNFa secretion. This is the case also for sulfasa-
lazine, an NF-kB inhibitor, since it results in full inhibition of
iNOS expression but only a modest downregulation of TNFa
production (data not shown). In addition, some flavonoids
(quercetin, luteolin and genistein) are more effective than
sulfasalazine in inhibiting TNFa production. Hence there must
be additional pathways involved in the mechanism of action
of flavonoids. The mitogen-activated protein kinases (MAPK)
family members are a group of serine/threonine kinases that
become activated downstream of TLR4 ligation by LPS in
b i o c h e m i c a l p h a r m a c o l o g y 7 2 ( 2 0 0 6 ) 1 0 1 0 – 1 0 2 1 1019
macrophages. Many studies have demonstrated that inhibition
of extracellular signal-regulated (ERK)-1/2, c-Jun N-terminal
Kinase (JNK) and p38 activation affects LPS-stimulated TNFa
production in macrophages [21,49,55]. Studies are underway to
determine the possible effect of flavonoids at this level.
Finally, since it is well established that the inflammatory
response may be resolved through the clearance of the
inflammatory cells or through the release of endogenous
anti-inflammatory mediators such as IL-10 and TGF-b [56,57],
we decided to analyze the effect of flavonoids in IL-10 secretion
induced by LPS in macrophages. We have demonstrated for the
first time to our knowledge that luteolin and quercetin
stimulate LPS-induced IL-10 secretion. It is interesting to note
that this effect vanishes at higher concentrations. This may be
due to post-transcriptional effects of the flavonoids, since it is
well established that IL-10 can be regulated by post-transcrip-
tional mechanisms in macrophages [58]. Alternatively, flavo-
noids may be acting as prooxidative molecules at high
concentrations. The differential effect observed with these
compounds compared with apigenin and the other flavonoids
tested constitutes additional evidence that NF-kB is not the only
signaling pathway affected by flavonoids. The possible effect of
flavonoids on p38 MAPK, which is involved in the regulation of
IL-10 expression, is being studied [59].
Since flavonoids are particularly known for their antiox-
idative/radical scavenging properties, which may be partly
related to their anti-inflammatory actions and specifically with
inhibition of the NF-kB pathway, it is interesting to consider this
aspect in relation to the observed effects in this study. The main
determinants of antioxidative capacity have been classically
considered to be the presence of a catechol group in the B ring,
the presence of a 3-OH group, and the C2–C3 double bond [60].
Thus there is a partial correlation with the structure–activity
relationship of the flavonoids as inhibitors of iNOS and TNFa
induction in macrophages, since quercetin and luteolin are the
most active compounds in both categories. However, kaemp-
ferol is an inferior inhibitor despite having substantial
antioxidative activity, while apigenin is a poor antioxidant
with high in vitro anti-inflammatory effects [61]. Thus, we
suggest that antioxidative properties probably do not play a
primary role in these flavonoid mechanisms.
Considered together, our results suggest that luteolin and
quercetin may be the best flavonoid candidates to provide
anti-inflammatory relief in vivo because of their inhibitory
effects on TNFa and iNOS expression coupled to the
enhancement of IL-10 release. However, the pharmacokinetic
characteristics of flavonoids have to be considered before
these agents may be used successfully in vivo. Most flavonoids
are found glycosylated in nature and cannot be absorbed by
the intestinal epithelium in that form [62]. Enzymatic
hydrolysis of the glycosides by the intestinal flora yields free
flavonoids that are absorbed efficiently. Thus it is likely that
these flavonoids must be administered in glycosidic form in
order to be applied as anti-inflammatory agents.
Acknowledgements
This work was supported by grants from the Spanish Ministry
of Science and Technology (SAF2002-02592 and SAF2005-
03199), from the Instituto de Salud Carlos III (PI051625) and
from the Junta de Andalucı́a (CTS-164). MC is a member of the
‘‘Juan de la Cierva’’ program, Ministerio de Ciencia y
Tecnologı́a, Spain. IB is a recipient of a pre-doctoral fellowship
from Fundación Ramón Areces, Madrid, Spain. EB is a recipient
of a pre-doctoral fellowship from the Ministerio de Ciencia y
Tecnologı́a, Spain.
r e f e r e n c e s
[1] Hertog MG, Hollman PC, Katan MB, Kromhout D. Intake ofpotentially anticarcinogenic flavonoids and theirdeterminants in adults in The Netherlands. Nutr Cancer1993;20:21–9.
[2] Bravo L. Polyphenols: chemistry, dietary sources,metabolism, and nutritional significance. Nutr Rev1998;56:317–33.
[3] Modolo LV, Cunha FQ, Braga MR, Salgado I. Nitric oxidesynthase-mediated phytoalexin accumulation in soybeancotyledons in response to the Diaporthe phaseolorum f. sp.meridionalis elicitor. Plant Physiol 2002;130:1288–97.
[4] Scuro LS, Simioni PU, Grabriel DL, Saviani EE, Modolo LV,Tamashiro WM, et al. Suppression of nitric oxideproduction in mouse macrophages by soybean flavonoidsaccumulated in response to nitroprusside and fungalelicitation. BMC Biochem 2004;5:5.
[5] Middleton Jr E, Kandaswami C, Theoharides TC. The effectsof plant flavonoids on mammalian cells: implications forinflammation, heart disease, and cancer. Pharmacol Rev2000;52:673–751.
[6] Camuesco D, Comalada M, Rodriguez-Cabezas ME, Nieto A,Lorente MD, Concha A, et al. The intestinal anti-inflammatory effect of quercitrin is associated with aninhibition in iNOS expression. Br J Pharmacol 2004;143:908–18.
[7] Fatehi M, Anvari K, Fatehi-Hassanabad Z. The beneficialeffects of protein tyrosine kinase inhibition on thecirculatory failure induced by endotoxin in the rat. Shock2002;18:450–5.
[8] Hendriks JJ, Alblas J, van der Pol SM, van Tol EA, DijkstraCD, de Vries HE. Flavonoids influence monocytic GTPaseactivity and are protective in experimental allergicencephalitis. J Exp Med 2004;200:1667–72.
[9] Kawaguchi K, Kikuchi S, Hasunuma R, Maruyama H,Yoshikawa T, Kumazawa Y. A citrus flavonoid hesperidinsuppresses infection-induced endotoxin shock in mice. BiolPharm Bull 2004;27:679–83.
[10] Singh B, Read S, Asseman C, Malmstrom V, Mottet C,Stephens LA, et al. Control of intestinal inflammation byregulatory T cells. Immunol Rev 2001;182:190–200.
[11] De Rycke L, Vandooren B, Kruithof E, De Keyser F, Veys EM,Baeten D. Tumor necrosis factor alpha blockade treatmentdown-modulates the increased systemic and localexpression of toll-like receptor 2 and toll-like receptor 4 inspondylarthropathy. Arthritis Rheum 2005;52:2146–58.
[12] Kamphuis S, Kuis W, de Jager W, Teklenburg G, Massa M,Gordon G, et al. Tolerogenic immune responses to novel Tcell epitopes from heat-shock protein 60 in juvenileidiopathic arthritis. Lancet 2005;366:50–6.
[13] Martin M, Rehani K, Jope RS, Michalek SM. Toll-likereceptor-mediated cytokine production is differentiallyregulated by glycogen synthase kinase 3. Nat Immunol2005;6:777–84.
[14] Celada A, Nathan C. Macrophage activation revisited.Immunol Today 1994;15:100–2.
b i o c h e m i c a l p h a r m a c o l o g y 7 2 ( 2 0 0 6 ) 1 0 1 0 – 1 0 2 11020
[15] Stanley ER, Berg KL, Einstein DB, Lee PS, Pixley FJ, Wang Y,et al. Biology and action of colony-stimulating factor-1. MolReprod Dev 1997;46:4–10.
[16] Boehm JE, Chaika OV, Lewis RE. Anti-apoptotic signaling bya colony-stimulating factor-1 receptor/insulin receptorchimera with a juxtamembrane deletion. J Biol Chem1998;273:7169–76.
[17] Nogai A, Siffrin V, Bonhagen K, Pfueller CF, Hohnstein T,Volkmer-Engert R, et al. Lipopolysaccharide injectioninduces relapses of experimental autoimmuneencephalomyelitis in nontransgenic mice via bystanderactivation of autoreactive CD4+ cells. J Immunol2005;175:959–66.
[18] Xagorari A, Papapetropoulos A, Mauromatis A, EconomouM, Fotsis T, Roussos C. Luteolin inhibits an endotoxin-stimulated phosphorylation cascade and proinflammatorycytokine production in macrophages. J Pharmacol Exp Ther2001;296:181–7.
[19] Yamamoto Y, Gaynor RB. IkappaB kinases: key regulatorsof the NF-kappaB pathway. Trends Biochem Sci 2004;29:72–9.
[20] Blonska M, Czuba ZP, Krol W. Effect of flavone derivativeson interleukin-1beta (IL-1beta) mRNA expression and IL-1beta protein synthesis in stimulated RAW 264.7macrophages. Scand J Immunol 2003;57:162–6.
[21] Kim HK, Cheon BS, Kim YH, Kim SY, Kim HP. Effects ofnaturally occurring flavonoids on nitric oxide production inthe macrophage cell line RAW 264.7 and their structure–activity relationships. Biochem Pharmacol 1999;58:759–65.
[22] Kim SJ, Park H, Kim HP. Inhibition of nitric oxide productionfrom lipopolysaccharide-treated RAW 264.7 cells bysynthetic flavones: structure–activity relationship andaction mechanism. Arch Pharm Res 2004;27:937–43.
[23] Liang YC, Huang YT, Tsai SH, Lin-Shiau SY, Chen CF, Lin JK.Suppression of inducible cyclooxygenase and induciblenitric oxide synthase by apigenin and related flavonoids inmouse macrophages. Carcinogenesis 1999;20:1945–52.
[24] Blonska M, Bronikowska J, Pietsz G, Czuba ZP, Scheller S,Krol W. Effects of ethanol extract of propolis (EEP) and itsflavones on inducible gene expression in J774A.1macrophages. J Ethnopharmacol 2004;91:25–30.
[25] Comalada M, Xaus J, Sanchez E, Valledor AF, Celada A.Macrophage colony-stimulating factor-, granulocyte–macrophage colony-stimulating factor-, or IL-3-dependentsurvival of macrophages, but not proliferation, requires theexpression of p21(Waf1) through the phosphatidylinositol3-kinase/Akt pathway. Eur J Immunol 2004;34:2257–67.
[26] Kang BY, Chung SW, Im SY, Choe YK, Kim TS. Sulfasalazineprevents T-helper 1 immune response by suppressinginterleukin-12 production in macrophages. Immunology1999;98:98–103.
[27] Pan MH, Lin-Shiau SY, Lin JK. Comparative studies on thesuppression of nitric oxide synthase by curcumin and itshydrogenated metabolites through down-regulation ofIkappaB kinase and NFkappaB activation in macrophages.Biochem Pharmacol 2000;60:1665–76.
[28] Celada A, Gray PW, Rinderknecht E, Schreiber RD. Evidencefor a gamma-interferon receptor that regulatesmacrophage tumoricidal activity. J Exp Med 1984;160:55–74.
[29] Comalada M, Cardo M, Xaus J, Valledor AF, Lloberas J,Ventura F, et al. Decorin reverses the repressive effect ofautocrine-produced TGF-beta on mouse macrophageactivation. J Immunol 2003;170:4450–6.
[30] Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH,Provenzano MD, et al. Measurement of protein usingbicinchoninic acid. Anal Biochem 1985;150:76–85.
[31] Chen D, Daniel KG, Chen MS, Kuhn DJ, Landis-Piwowar KR,Dou QP. Dietary flavonoids as proteasome inhibitors and
apoptosis inducers in human leukemia cells. BiochemPharmacol 2005;69:1421–32.
[32] Chowdhury SA, Kishino K, Satoh R, Hashimoto K, KikuchiH, Nishikawa H, et al. Tumor-specificity and apoptosis-inducing activity of stilbenes and flavonoids. AnticancerRes 2005;25:2055–63.
[33] Cui YY, Xie H, Qi KB, He YM, Wang JF. Effects of Pinusmassoniana bark extract on cell proliferation and apoptosisof human hepatoma BEL-7402 cells. World J Gastroenterol2005;11:5277–82.
[34] Korhonen R, Lahti A, Kankaanranta H, Moilanen E. Nitricoxide production and signaling in inflammation. Curr DrugTargets Inflamm Allergy 2005;4:471–9.
[35] Reinders CI, Herulf M, Ljung T, Hollenberg J, Weitzberg E,Lundberg JO, et al. Rectal mucosal nitric oxide indifferentiation of inflammatory bowel disease and irritablebowel syndrome. Clin Gastroenterol Hepatol 2005;3:777–83.
[36] Xaus J, Comalada M, Valledor AF, Lloberas J, Lopez-SorianoF, Argiles JM, et al. LPS induces apoptosis in macrophagesmostly through the autocrine production of TNF-alpha.Blood 2000;95:3823–31.
[37] Beinke S, Ley SC. Functions of NF-kappaB1 and NF-kappaB2in immune cell biology. Biochem J 2004;382:393–409.
[38] Neurath MF, Becker C, Barbulescu K. Role of NF-kappaB inimmune and inflammatory responses in the gut. Gut1998;43:856–60.
[39] Coombes JL, Robinson NJ, Maloy KJ, Uhlig HH, Powrie F,Regulatory. T cells and intestinal homeostasis. ImmunolRev 2005;204:184–94.
[40] Kishore R, Tebo JM, Kolosov M, Hamilton TA. Cutting edge:clustered AU-rich elements are the target of IL-10-mediatedmRNA destabilization in mouse macrophages. J Immunol1999;162:2457–61.
[41] de Waal Malefyt R, Abrams J, Bennett B, Figdor CG, de VriesJE. Interleukin 10 (IL-10) inhibits cytokine synthesis byhuman monocytes: an autoregulatory role of IL-10produced by monocytes. J Exp Med 1991;174:1209–20.
[42] Xaus J, Comalada M, Valledor AF, Cardo M, Herrero C, SolerC, et al. Molecular mechanisms involved in macrophagesurvival, proliferation, activation or apoptosis.Immunobiology 2001;204:543–50.
[43] Jung M, Sabat R, Kratzschmar J, Seidel H, Wolk K,Schonbein C, et al. Expression profiling of IL-10-regulatedgenes in human monocytes and peripheral bloodmononuclear cells from psoriatic patients during IL-10therapy. Eur J Immunol 2004;34:481–93.
[44] Lin JK. Cancer chemoprevention by tea polyphenolsthrough modulating signal transduction pathways. ArchPharm Res 2002;25:561–71.
[45] Karin M, Lin A. NF-kappaB at the crossroads of life anddeath. Nat Immunol 2002;3:221–7.
[46] Fotsis T, Pepper MS, Aktas E, Breit S, Rasku S, AdlercreutzH, et al. Flavonoids, dietary-derived inhibitors of cellproliferation and in vitro angiogenesis. Cancer Res1997;57:2916–21.
[47] Verbeek R, Plomp AC, van Tol EA, van Noort JM. Theflavones luteolin and apigenin inhibit in vitro antigen-specific proliferation and interferon-gamma production bymurine and human autoimmune T cells. BiochemPharmacol 2004;68:621–9.
[48] Constantinou A, Mehta R, Runyan C, Rao K, Vaughan A,Moon R. Flavonoids as DNA topoisomerase antagonists andpoisons: structure–activity relationships. J Nat Prod1995;58:217–25.
[49] Comalada M, Xaus J, Valledor AF, Lopez-Lopez C,Pennington DJ, Celada A. PKC epsilon is involved in JNKactivation that mediates LPS-induced TNF-alpha, whichinduces apoptosis in macrophages. Am J Physiol CellPhysiol 2003;285:C1235–4.
b i o c h e m i c a l p h a r m a c o l o g y 7 2 ( 2 0 0 6 ) 1 0 1 0 – 1 0 2 1 1021
[50] Kempuraj D, Madhappan B, Christodoulou S, Boucher W,Cao J, Papadopoulou N, et al. Flavonols inhibitproinflammatory mediator release, intracellular calciumion levels and protein kinase C theta phosphorylation inhuman mast cells. Br J Pharmacol 2005;145:934–44.
[51] Chen JC, Ho FM, Pei-Dawn Lee Chao, Chen CP, Jeng KC, HsuHB, et al. Inhibition of iNOS gene expression by quercetin ismediated by the inhibition of IkappaB kinase, nuclearfactor-kappa B and STAT1, and depends on hemeoxygenase-1 induction in mouse BV-2 microglia. Eur JPharmacol 2005;521:9–20.
[52] Kim HR, Pham HT, Ziboh VA. Flavonoids differentiallyinhibit guinea pig epidermal cytosolic phospholipase A2.Prostag Leukotr Essent Fatty Acids 2001;65:281–6.
[53] Pignatelli P, Pulcinelli FM, Celestini A, Lenti L, Ghiselli A,Gazzaniga PP, et al. The flavonoids quercetin and catechinsynergistically inhibit platelet function by antagonizing theintracellular production of hydrogen peroxide. Am J ClinNutr 2000;72:1150–5.
[54] Hashimoto T, Kihara M, Yokoyama K, Fujita T, Kobayashi S,Matsushita K, et al. Lipoxygenase products regulate nitricoxide and inducible nitric oxide synthase production ininterleukin-1beta stimulated vascular smooth muscle cells.Hypertens Res 2003;26:177–84.
[55] Xagorari A, Roussos C, Papapetropoulos A. Inhibition ofLPS-stimulated pathways in macrophages by the flavonoidluteolin. Br J Pharmacol 2002;136:1058–64.
[56] Di Giacinto C, Marinaro M, Sanchez M, Strober W, BoirivantM. Probiotics ameliorate recurrent Th1-mediated murine
colitis by inducing IL-10 and IL-10-dependent TGF-beta-bearing regulatory cells. J Immunol 2005;174:3237–46.
[57] Torre D, Tambini R, Aristodemo S, Gavazzeni G, Goglio A,Cantamessa C, et al. Anti-inflammatory response of IL-4,IL-10 and TGF-beta in patients with systemic inflammatoryresponse syndrome. Mediat Inflamm 2000;9:193–5.
[58] Nemeth ZH, Lutz CS, Csoka B, Deitch EA, Leibovich SJ,Gause WC, et al. Adenosine augments IL-10 production bymacrophages through an A2B receptor-mediatedposttranscriptional mechanism. J Immunol 2005;175:8260–70.
[59] Foey AD, Parry SL, Williams LM, Feldmann M, Foxwell BM,Brennan FM. Regulation of monocyte IL-10 synthesis byendogenous IL-1 and TNF-alpha: role of the p38 and p42/44mitogen-activated protein kinases. J Immunol1998;160:920–8.
[60] Silva MM, Santos MR, Caroco G, Rocha R, Justino G, Mira L.Structure–antioxidant activity relationships of flavonoids: are-examination. Free Radic Res 2002;36:1219–27.
[61] Nemeth K, Plumb GW, Berrin JG, Juge N, Jacob R, Naim HY,et al. Deglycosylation by small intestinal epithelial cellbeta-glucosidases is a critical step in the absorption andmetabolism of dietary flavonoid glycosides in humans. EurJ Nutr 2003;42:29–42.
[62] Comalada M, Camuesco D, Sierra S, Ballester I, Xaus J,Galvez J, et al. In vivo quercitrin anti-inflammatory effectinvolves release of quercetin, which inhibits inflammationthrough down-regulation of the NF-kB pathway. Eur JImmunol 2005;35:584–92.
Inhibition of pro-inflammatory markers in primary �bone marrow-derived mouse macrophages by �naturally occurring flavonoids: Analysis of the �structure-activity relationshipIntroductionMaterials and methodsReagentsMacrophage cultureProliferation assayDetermination of TNFα and IL-10 concentrationProtein extraction and Western blot analysisDetermination of NO productionAnalysis of gene expression by RT-PCRStatistical analysis
ResultsEffects of flavonoids on M-CSF-macrophage proliferation and viabilityEffect of flavonoids on TNFα release by LPS-activated macrophagesEffect of flavonoids on iNOS expression and NO release by LPS-activated macrophagesEffect of flavonoids on LPS-induced IκB-α phosphorylationLuteolin and quercetin induce IL-10 secretion by LPS-activated macrophages
DiscussionAcknowledgementsReferences